PROJECT SUMMARY Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma, a subset of multicentric Castleman's disease, and Kaposi's sarcoma, the most common malignancy in HIV/AIDS patients. KSHV ORF45, an immediate-early gene and tegument protein, activates the cellular kinase RSK, which is essential for optimal lytic gene expression and progeny virion production. The precise role(s) of ORF45-induced RSK activation that contribute to efficient KSHV lytic replication remain to be determined. The long-term goal is to elucidate the mechanism(s) by which KSHV facilitates efficient viral gene expression and productive viral replication by appropriating the host cellular machinery, and how this contributes to KSHV pathogenesis. The immediate objective is to define the roles of ORF45-activated RSK throughout KSHV lytic replication, including contributions of ORF45/RSK substrates to viral epigenomic modifications, transcription and translation. The central hypothesis is that KSHV ORF45-activated RSK alters the activities of several diverse substrates during KSHV lytic replication, resulting in the regulation of epigenomic modifications, transcription and translation. This hypothesis was formulated upon reviewing the primary literature and preliminary data produced by the applicant. The rationale that underlies the proposed research is that it will allow for hypothesis-driven approaches to reveal a mechanistic explanation for the apparent regulation of epigenomic modifications and viral/cellular gene expression by KSHV. The central hypothesis will be tested by pursuing the following specific aims: 1) Determine the roles of KSHV ORF45-activated RSK in the regulation of histone modifications and the consequences on viral and cellular transcription; and 2) Characterize the effects of ORF45-induced RSK activation on viral and cellular translation. To achieve these aims, a system for efficient and inducible KSHV lytic reactivation will be employed. For the first aim, chromatin immunoprecipitation and next-generation sequencing will be used to accurately measure the effect(s) of both KSHV lytic replication and ORF45 activation of RSK on transcriptional regulation. The second aim will make use of various approaches, including an innovative ribosome profiling technique, to elucidate the functional significance of ORF45- activated RSK with regard to translational control. The expected contributions of the proposed research are to corroborate roles for ORF45-activated RSK in critical regulatory processes during KSHV lytic replication. These contributions will be significant because they will further the understanding of KSHV-induced dysregulation of the host cell signaling pathways, which may be useful in the development of novel treatments for KSHV-related diseases.